Influences Of Selenium On Several Toxic Metabolites Produced By Two Endophytic Fungal Strains Isolated From KBD-prevalent Areas

Kashin-Beck Disease(KBD)is a chronical and local osteoarthropathy,which seriously affects the health of human,but the pathogenesis of KBD is not yet fully understood.In our recent researches,we found that selenium in suitable concentration could inhibit the ability of toxin production of the tested strains,thus,reduced the toxicity of fermented product to chondrocyte cultured in vitro.The influence of mycotoxin to chondrocyte was likely to be a result of the combined action of some kinds of toxins.This study was carried out on the basis of the above work.The experimental stains of Fusarium poae and Alternaria alternate were isolated from wheat in a KBD-prevalent region in Qinghai province.Their fermented products were analyzed by organic solvent extraction,silica gel column chromatography,thin-layer chromatography(TLC),high-performance liquid chromatography(HPLC)and liquid chromatography mass spectrometry(LC-MS).Besides,the influence of toxin combination and selenium-DAS to chondrocyte of rabbit knee were explored.Cell viability was detected by MTT assay.The quantity of collagen II was measured by western blotting and immunohistochemical.The quantity of aggrecan was measured by toluidine blue staining and enzyme linked immunosorbent assay(ELASA).The mRNA expression of collagen II and aggrecan was detected by semiquantitative RT-PCR and real-time quantification PCR(qPCR).The results showed that the yield of T-2 toxin,DAS and AME was the lowest when selenium concentrations added in the fermentation medium were 20,20 and 5mg/L,respectively.Besides,compared to the treatment with no-selenium,all of the toxin yields with selenium were lower than that with no selenium.The results of the influence of toxin combination showed that the damage effect from high to low of the single toxin on chondrocyte in the same concentration were T-2 toxin,DAS and AME.The damage effects from high to low of the toxin combination on chondrocyte were T-2 toxin with DAS,T-2 toxin with AME and AME with DAS.Furthermore,the toxin combination which included AME showed less inhibited effect to cell viability and mRNA expression of collagen Ⅱ and aggrecan compared to the single toxin.The results showed that DAS had more inhibitory effect than selenium on cell viability,mRNA expression of collagen Ⅱ and aggrecan.The combination of 0.05 mg/L selenium and 0.001 mg/L DAS showed the maximum protective effect to cell.These results implied that selenium could reduce the cell damage from DAS in a suitable concentration.In summary,T-2 toxin,DAS and AME yields in the fermentative product was reduced by the addition of selenium to the fungal culture medium in varying degrees.Selenium could reduce the cell damage induced by DAS in a suitable concentration.Moreover,the combination of different mycotoxins could showed some synergistic effect on the damage of chondrocyte.The results suggest that starting with the relationship between fungal metabolism and environmental factors,it may be possible to provide a new clue into the causes of the KBD.